Rev. Med. Chir. Soc. Med. Nat., Iaşi – 2012 – vol. 116, no. 4 PHARMACY ORIGINAL PAPERS EXPERIMENTAL RESEARCHES ON ACUTE TOXICITY OF A BIDENS TRIPARTITA EXTRACT IN MICE - PRELIMINARY INVESTIGATIONS R. B. Sandu1, L. Tarţău2, A. Miron3, M. Zagnat4, C. M. Ghiciuc2, C. E. Lupuşoru2 University of Medicine and Pharmacy ”Grigore T. Popa” - Iasi 1. Ph.D. student Faculty of Medicine 2. Discipline of Pharmacology 3. Faculty of Pharmacy 4. Faculty of Medical Bioengineering EXPERIMENTAL RESEARCHES ON ACUTE TOXICITY OF A BIDENS TRIPARTITA EX- TRACT IN MICE - PRELIMINARY INVESTIGATIONS (Abstract): Plants take up an im- portant place in traditional medicine and scientific research confirmed properties about their use as alternative therapy. Bidens tripartita, commonly known as Three-lobe Beggarticks, Three-part Beggarticks, Trifid Bur-marigold, is a flowering plant in the genus Bidens, family Compositae, subfamily Asteroideae. Evaluation of the chemical composition of this plant has revealed the presence of flavonoids, xanthophylls, volatile oil, acetylene and polyacetylene, sterols, aurones, chalcones, caffeine and tannins. Aim: Theoretical data investigation regarding Bidens tripartita plant and experimental researches on acute toxicity of an original extract in mice. Material and methods: The vegetal product of Bidens tripartita used for study was ob- tained by maceration and extraction in alcohol, and its chemical composition was determined. Acute toxicity of the alcoholic extract of Bidens tripartita was assessed by median lethal dose (LD50) calculation, using a limit dose test of up- and- down procedure at a limit dose of 2000mg/kbw after intraperitoneal administration in mice. Results: In the alcoholic extract of Bidens tripartita different active principles were identified: tannins, anthracene derivatives, triterpenes, coumarins, antocyanosides. The toxicity of plant product was evaluated by differ- ent characteristic signs for the mouse which can be retained as toxicity elements of the extract. Using the intraperitoneal route, the animals showed dose-dependent signs of toxicity, ranging from lack of appetite, depression, immobility and respiratory distress to death. Single-dose in- traperitoneal LD50 value of the alcoholic Bidens tripartita extract in mice was 4038 mg/kg. No macroscopic changes were seen in the organs of mice that died following extract administra- tion. Histopathological lesions were not found in all examined organs. Conclusions: The ob- tained LD50 value classifies the study plant extract as slightly toxic according to Hodge and Sterner toxicity scale. We determined the low toxic dose at a rate of 4038 mg of body weight for the alcoholic extract of this medicinal plant. These results suggest that the alcoholic extract of Bidens tripartita is relatively safe toxicologically when administered intraperitoneally, and this product could be used with some degree of safety to continue the investigation for in vivo biocompatibility evaluation. Keywords: BIDENS TRIPARTITA, LETHAL DOSE 50, MICE. Adaptogens are naturally occurring sub- which appear to increase body ability to stances found in rare plants and herbs, adapt to stress and changing situations (1). 1230 Experimental researches on acute toxicity of a Bidens tripartita extract The exact mechanism through which an coholic extract of Bidens tripartita plant, adaptogen works is unclear so far, as it establishing its composition and determin- seems to be caused by the effects of differ- ing the lethal dose 50% (LD50) after acute ent chemical compounds. In general, adap- intraperitoneal administration in mice. togens are all potent antioxidants and they Collection and processing of plant ma- are reported to improve endurance and terial. The plant material for the study was produce effects associated with stress re- represented by an alcoholic extract of duction, for instance, improved sleep and Bidens tripartita L. plant. Plants were col- enhanced physical performances (2). lected during flowering stage in July and The main effects of adaptogens are rep- August 2009, 2010, 2011 from the Ciric resented by: an increased availability of area, Iasi district, where it grows naturally. energy during the day, a reduction of stress This medicinal plant species was taxonom- feelings, increased endurance, greater men- ically identified and authenticated by bo- tal alertness, and deep and restful sleep (3). tanical specialists. Also, adaptogens significantly accelerate The vegetal product of Bidens tripartita the recovery process after illness. They can used for the study was obtained by macera- have individual effects, depending on gen- tion and extraction in alcohol, and its eral health, constitution, specific nutrient chemical composition was determined. deficiencies, and medical conditions pre- The plants were washed in water and sent in an individual (4). dried in laboratory using continuous ventila- Bidens tripartita (B. tripartita), common- tion away from sun light and dust. Then, ly known as Three-lobe Beggarticks, Three- they were cut into smaller pieces and ground part Beggarticks, Trifid Bur-marigold, is a into a coarse powder with a blender. This flowering plant in the genus Bidens, family procedure ensures proper penetration of the Compositae, subfamily Asteroideae. It is extracting solvent into the cell to facilitate found along streams and ditches, and flowers the release of the flower’s active ingredi- during late summer and autumn. ents. The vegetable product was kept in Literature data reported that B. triparti- closed plastic bag. The powder was dis- ta contains a significant amount of flavo- solved in absolute chloroform (96%) and re- noids, xanthophylls, volatile oil, acetylene extracted seven (times (repeated macera- and polyacetylene, sterols, aurones, chal- tion). The suspension was filtered by filter cones, caffeine and tannins (5). paper and dried using a Rota vapour. Then, In traditional medicine, infusions of B. a jet air was used to remove the solvent tripartita are widely used in the treatment smell. After complete dryness, the product of catarrhal rhinitis, angina, acute respira- was extracted by the addition of ethanol tory infection, and as an anti-inflammatory seven times (repeated maceration) following in colitis and gout (6). It also proved anti- the same procedure of evaporation. Finally, septic, anti-inflammatory, antioxidant, as- the extract was weighed by digital balance, tringent, diuretic, febrifuge, narcotic, seda- stored in bottles and kept in the laboratory tive and sudorific effects (1,7). refrigerator for further research (8). Animals. Swiss white male mice (30-40 MATERIAL AND METHODS g) were used. The animals were housed The study consisted in obtaining an al- under standard laboratory conditions (rela- 1231 R. B. Sandu et al. tive humidity 55-65%, room temperature The toxicity of plant product was evalu- 23.0±2.0°C and 12 hours light: dark cycle ated by different characteristic signs for the (lights on at 6:00 a.m.)). The animals were mouse which can be retained as toxicity fed with standard diet and water ad libitum, elements of the extract. Using the intraperi- except during the time of experiments. toneal route, the animals showed dose- Before the experiment, mice were placed dependent signs of toxicity, ranging from on a raised wire mesh, under a clear plastic lack of appetite, depression, immobility box and allowed 2 hours to acclimate to the and respiratory distress to death (11). testing room. The arithmetic method of Karber with Acute toxicity investigation. This study the following formula was used: was designed to evaluate the Bidens tripar- LDL50 = LD100 - ∑(axb) / n tita extract acute toxicity in vivo, after intraperitoneal administration in mice. a = the difference between two succes- Median lethal dose (LD50) is the dose, sive doses of administered substance given all at once, which causes the death of b = the average number of dead animals half the number of test animals. LD50 is in two successive groups one way to measure the short-term toxic n = the number of animals in a group, potential of a product (9). Establishing of DL100 = lethal dose 100% (represent- LD50 in lab animals is very important to ing the dose, given all at once, which caus- estimate the degree of acute toxicity of a es the death all test animals). tested substance (12). Graphic curve was realized with exper- The acute toxicity of the alcoholic imental data to have LD01, LD50, LD99. Bidens tripartita extract was assessed by Much more, two ratios: LD99/LD50 and LD50 calculation, using a limit dose test LD50/LD01 were calculated and compared. (up-and- down procedure) at a limit dose of The determined LD50 value allowed us 2000 mg/kg bw of each vegetal extract to find out the level of toxicity of the plant after intraperitoneal administration in mice product on the toxicity scale suggested by (five animals per group) (OECD-OCDE WHO/IPCS (2002) representing an adapta- 425 Guide). tion of the Hodge and Sterner scale (1980) LD50 determination of Bidens tripartita (1). extracts was estimated where all doses Data were statistically analyzed with were expressed in terms of extract weight/ SPSS software for Windows version 17.0 animal weight. Preliminary experiments and ANOVA one-way method. The results were done to determine the minimal dose were expressed as mean of LD01, LD50 and that kills all animals (LD100) and the max- LD99 ± S.E.M. (standard error of mean). imal dose that fails to kill any animal. Sev- Experimental protocols were imple- eral doses at equal logarithmic intervals mented according to recommendations of were chosen in between these two doses, the “Grigore T. Popa” University Commit- each dose being injected intraperitoneally tee for Research and Ethical Issues. Each in a group of five animals. The mice were animal was used only once, and the dura- observed for 14 days and symptoms of tion of the experiments was kept as short as toxicity and mortality rates in each group possible. For ethical reasons, all the ani- were recorded and LD50 was calculated. mals were sacrificed at the end of the ex- 1232 Experimental researches on acute toxicity of a Bidens tripartita extract periment (13).